1. Field of Art
The present invention relates to the biochemical arts, particularly to conjugates with immunoglobulin Fc domains.
2. Discussion of Related Art
The immunoglobulin Fc domain has found widespread use as a carrier protein for a variety of therapeutic and diagnostic molecules. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas a Fab is short-lived. (Capon et al. (1989), Nature 337: 525-31). When constructed together with a therapeutic protein or peptide, an Fc domain can provide longer half-life, or can incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id.
Numerous fusions of proteins and peptides have been engineered at either the amino- or carboxy-termini. Also, a variety of enzymes and synthetic reporter molecules have been chemically conjugated to the side chains of non-terminal amino acids as well as the derivatized carbohydrate moieties of the Fc domain. Further, several polymers, such as polyethylene glycol (PEG) have been conjugated to the Fc domain for the purpose of improved half-life in vivo and reduced immunogenicity.
The success of the drug Enbrel® (etanercept) brought to fruition the promise of therapeutic agents modified with the constant domain of an antibody. Table 1 summarizes several examples of the use of Fc fusion proteins known in the art.
TABLE 1Fc fusion with therapeutic proteinsTherapeuticForm of FcFusion partnerimplicationsReferenceIgG1N-terminus ofHodgkin's disease;U.S. Pat. No.CD30-Lanaplastic lymphoma;5,480,981T-cell leukemiaMurine Fcγ2aIL-10Anti-inflammatory;Zheng et al., (1995), J.transplant rejectionImmunol.154: 5590-600IgG1TNF receptorSeptic shockFisher et al. (1996), N.Engl. J. Med. 334:1697-1702; Van Zee,K. et al. (1996), J.Immunol. 156: 2221-30IgG, IgA,TNF receptorInflammation,U.S. Pat. No.IgM, or IgEautoimmune disorders5,808,029, issued(excludingSep. 15, 1998the firstdomain)IgG1CD4 receptorAIDSCapon et al. (1989),Nature 337: 525-31IgG1, IgG3N-terminus ofAnti-cancer, antiviralHarvill et al. (1995),IL-2Immunotech. 1: 95-105IgG1C-terminus ofosteoarthritis; boneWO 97/23614,OPGdensitypublished Jul. 3, 1997IgG1N-terminus ofAnti-obesityWO 98/28427, filedleptinDec. 11, 1997Human IgCTLA-4Autoimmune disordersLinsley (1991), J. Exp.C 1Med. 174: 561-9
A more recent development is fusion of randomly generated peptides with the Fc domain. See U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 to Feige et al. (incorporated by reference in its entirety). Such molecules have come to be known as “peptibodies.” They include one or more peptides linked to the N-terminus, C-terminus, amino acid side chains, or to more than one of these sites. Peptibody technology enables design of therapeutic agents that incorporate peptides that target one or more ligands or receptors, tumor-homing peptides, membrane-transporting peptides, and the like. Peptibody technology has proven useful in design of a number of such molecules, including linear and disulfide-constrained peptides, “tandem peptide multimers” (i.e., more than one peptide on a single chain of an Fc domain). See, for example, U.S. Pat. No. 6,660,843; U.S. Pat. App. No. 2003/0195156 A1, published Oct. 16, 2003 (corresponding to WO 02/092620, published Nov. 21, 2002); U.S. Pat. App. No. 2003/0176352, published Sep. 18, 2003 (corresponding to WO 03/031589, published Apr. 17, 2003); U.S. Ser. No. 09/422,838, filed Oct. 22, 1999 (corresponding to WO 00/24770, published May 4, 2000); U.S. Pat. App. No. 2003/0229023, published Dec. 11, 2003; WO 03/057134, published Jul. 17, 2003; U.S. Pat. App. No. 2003/0236193, published Dec. 25, 2003 (corresponding to PCT/US04/010989, filed Apr. 8, 2004); U.S. Ser. No. 10/666,480, filed Sep. 18, 2003 (corresponding to WO 04/026329, published Apr. 1, 2004), U.S. Patent App. No. 2006/0140934, published Jun. 29, 2006 (corresponding to WO 2006/036834, published Apr. 4, 2006), each of which is hereby incorporated by reference in its entirety. The art would benefit from further technology enabling such rational design of polypeptide therapeutic agents.
Conventional approaches for chemical conjugation to the immunoglobulin Fc domain include random coupling to naturally occurring primary amines such as lysine and the amino-terminus or carboxylic acids such as glutamic acid, aspartic acid and the carboxy terminus. Alternatively, semi-selective site-specific coupling may be achieved through N-terminal conjugation under appropriate conditions, or derivatized carbohydrates as found on Fc proteins isolated from eukaryotic sources, or by partial reduction and coupling of native cysteine residues. (E.g., Kim et al., A pharmaceutical composition comprising an immunoglobulin Fc region as a carrier, WO 2005/047337). While each of these approaches has been applied successfully, they typically suffer from varying degrees of conjugate heterogeneity, relatively low yields and sometimes, significant losses in functional activity are also observed. The art would benefit from a process for selective, site-specific conjugation to the immunoglobulin Fc domain without significant loss in functional activity.